Ankrd7, a novel gene specifically expressed in Sertoli cells and its potential roles in Sertoli cell maturation

The somatic Sertoli cells play an essential role in testis determination and spermatogenesis by providing nutrition and structural support. In the current study, we report on the novel Ankrd7 gene that contains five ankyrin repeat domains. This gene was specifically expressed in Sertoli cells and was regulated in a maturation-dependent manner. Its expression was restricted to testicular tissue, and its mRNA could be detected in testes at as early as 14 dpp (days post partum) using RT-PCR analysis. In both testicular tissue sections and in vitro cultured Sertoli cells, the Ankrd7 protein was localized to the nucleus of the Sertoli cell. Immuno-histochemistry and immunocytochemistry investigations showed that the protein was detectable in testicular tissues at 20 dpp, at which time Sertoli cells were gradually differentiating into their mature cellular form. These results suggest that Ankrd7 is probably involved in the process of Sertoli cell maturation and in spermatogenesis.     

Influenza A virus protein PA-X suppresses host Ankrd17-mediated immune responses

Influenza A virus (IAV) PA-X is a critical ribonuclease protein involved in host cell shutoff but its role in modulating the host immune response to IAV infection remains to be addressed. In this study, host cellular proteins that directly interact with PA-X were screened to investigate the biological function of PA-X in the pathogenesis of IAV infection. The protein ankyrin repeat domain 17 (Ankrd17), a positive regulator of inflammatory responses via the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, was identified as a specific PA-X binding partner that preferred PA-X to the PA protein. The N-terminal ankyrin repeats of Ankrd17 are the key domain for the interaction with PA-X rather than PA, which is required for the function of Ankrd17 in elevating the host immune response. Using Ankrd17 knockout and overexpression, we confirmed that PA-X significantly affected the Ankrd17-mediated response to infection in host cells. Our data therefore reveal a novel function for PA-X in the regulation of innate immune pathways via the interaction between PA-X and Ankrd17.     

Suppression of nuclear Wnt signaling leads to stabilization of Rac1 isoforms

The Rac1 GTPase contains a functional nuclear localization signal (NLS) and destruction box sequence in the C-terminal polybasic region. It has been postulated that these two regulatory sequences may function together, enabling Rac1 to participate in nuclear signaling pathways that ultimately target it for degradation. We have previously shown that the NLS activity of Rac1 and the Rac1b splice variant is essential for Wnt pathway activation. In the present study, we demonstrate that suppression of nuclear Wnt signaling leads to stabilization of Rac1 protein. In addition, we show that Rac1b may be under proteasomal regulation. We propose that Rac1 and Rac1b levels are regulated by being targeted for degradation through a negative feedback loop initiated by Wnt signaling.     

Desumoylation of homeodomain-interacting protein kinase 2 (HIPK2) through the cytoplasmic-nuclear shuttling of the SUMO-specific protease SENP1

The modification of homeodomain-interacting protein kinase 2 (HIPK2) by small ubiquitin-like modifier 1 (SUMO-1) plays an important role in its targeting into the promyelocytic leukemia body, as well as in its differential interaction with binding partner, but the desumoylation of HIPK2 by SUMO-specific proteases is largely unknown. In this study, we show that HIPK2 is a desumoylation target for the SUMO-specific protease SENP1 that shuttles between the cytoplasm and the nucleus. Mutation analyses reveal that SENP1 contains the nuclear export sequence (NES) within the extreme carboxyl-terminal region, and SENP1 is exported to the cytoplasm in a NES-dependent manner. Sumoylated HIPK2 are deconjugated by SENP1 both in vitro and in cultured cells, and the desumoylation is enhanced either by the forced translocation of SENP1 into the nucleus or by the SENP1 NES mutant. Concomitantly, desumoylation induces dissociation of HIPK2 from nuclear bodies. These results demonstrate that HIPK2 is a target for SENP1 desumoylation, and suggest that the desumoylation of HIPK2 may be regulated by the cytoplasmic-nuclear shuttling of SENP1.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

Characterization of a nuclear compartment shared by nuclear bodies applying ectopic protein expression and correlative light and electron microscopy

To investigate the accessibility of interphase nuclei for nuclear body-sized particles, we analyzed in cultured cells from human origin by correlative fluorescence and electron microscopy (EM) the bundle-formation of Xenopus-vimentin targeted to the nucleus via a nuclear localization signal (NLS). Moreover, we investigated the spatial relationship of speckles, Cajal bodies, and crystalline particles formed by Mx1 fused to yellow fluorescent protein (YFP), with respect to these bundle arrays. At 37 degrees C, the nucleus-targeted, temperature-sensitive Xenopus vimentin was deposited in focal accumulations. Upon shift to 28 degrees C, polymerization was induced and filament arrays became visible. Within 2 h after temperature shift, arrays were found to be composed of filaments loosely embedded in the nucleoplasm. The filaments were restricted to limited areas of the nucleus between focal accumulations. Upon incubation at 28 degrees C for several hours, NLS vimentin filaments formed bundles looping throughout the nuclei. Speckles and Cajal bodies frequently localized in direct neighborhood to vimentin bundles. Similarly, small crystalline particles formed by YFP-tagged Mx1 also located next to vimentin bundles. Taking into account that nuclear targeted vimentin locates in the interchromosomal domain (ICD), we conclude that nuclear body-sized particles share a common nuclear space which is controlled by higher order chromatin organization.     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

Sequence, organization, and expression of the human FEM1B gene

The FEM-1 protein of Caenorhabditis elegans functions within the nematode sex-determination pathway. Two mouse homologs, encoded by the Fem1a and Fem1b genes, have been reported. We report here the characterization of a novel human gene, designated FEM1B, that is highly homologous to the mouse Fem1b gene. FEM1B encodes a protein, designated FEM1beta, that shows >99% amino acid identity to the corresponding mouse Fem1b protein, including 100% amino acid identity in the N-terminal ANK repeat domain. FEM1beta represents the first characterized human member of the FEM-1 protein family. The human and mouse genes show conservation of coding sequence and its intron/exon organization, flanking untranslated and genomic sequences, and expression pattern in adult tissues. These findings suggest that there may be evolutionary conservation of regulation and function between the mouse and human FEM1B genes.     

HPV16L1核浆运输的动力学过程

利用增强型绿色荧光蛋白(Enhancegreenflurenscentprotein,EGFP)标记不同的截短型HPV16L1蛋白(Humanpapillomavirustype16L1protein,HPV16L1),分析HPV16L1蛋白核定位信号(Nucleuslocationsignal,NLS)的作用。构建重组pFB-EGFP、pFB-EGFP-HPV16L1、pFB-EGFP-HPV16L1△NLS和pFB-EGFP-NLSHPV16L1p转移载体;在DH10Bac宿主菌内经Tn7转座子介导的同源重组后转染Sf-9细胞,获得重组Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS和Ac-EGFP-NLSHPV16L1杆状病毒,感染Sf-9昆虫细胞表达相应截短型HPV16L1融合蛋白;利用荧光显微镜和激光共聚焦显微镜观察不同融合蛋白的荧光特性和核浆转运动力学过程。结果发现Ac-EGFP杆状病毒感染的Sf-9细胞内明亮的绿色荧光均匀分布;重组Ac-EGFP-HPV16L1和Ac-EGFP-NLSHPV16L1杆状病毒感染的Sf-9细胞,明亮的绿色荧光主要位于细胞核内;重组Ac-EGFP-HPV16L1△NLS杆状病毒感染的Sf-9细胞,绿色荧光局限于细胞浆内,细胞核内无绿色荧光。说明HPV16L1蛋白羧基端的23个氨基酸(GKRKATPTTSSTSTTAKRKKRKL)具有完全核定位作用,能引导HPV16L1蛋白和EGFP突破核膜屏障进入Sf-9细胞核内。     

ANKRD26 and its interacting partners TRIO, GPS2, HMMR and DIPA regulate adipogenesis in 3T3-L1 cells

Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenesis and identify new targets for the modulation of adipogenesis.     

Ankrd2 is a modulator of NF-κB-mediated inflammatory responses during muscle differentiation

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3β as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.     

The cell senescence inducing gene product MORF4 is regulated by degradation via the ubiquitin/proteasome pathway

After undergoing several rounds of divisions normal human fibroblasts enter a terminally non-dividing state referred to as cellular or replicative senescence. We cloned MORF4 (mortality factor on human chromosome 4), as a cellular senescence inducing gene that caused immortal cells assigned to complementation group B for indefinite division to stop dividing. To facilitate analyses of this gene, which is toxic to cells at low levels, we obtained stable clones of HeLa cells expressing a tetracycline-induced MORF4 construct that could be induced by doxycycline in a dose-dependent manner. MORF4 induction resulted in reduced colony formation after 14 days of culture, as previously observed. We determined that MORF4 protein was unstable and that addition of the proteasome inhibitor MG132 resulted in the accumulation of the protein. Following removal of MG132 the protein was rapidly degraded. Subcellular fractionation following MG132 treatment demonstrated that the protein accumulates primarily in the cytoplasm with some amounts present in the nucleus. It is therefore possible that MORF4 protein, which escapes degradation in the cytoplasm, is transported to the nucleus where it is functional. The results suggest that levels of MORF4 in cells must be tightly controlled and one mechanism involves stability of the protein.     

植物细胞质介导GFP-NLS蛋白入核转运的HeLa细胞系统的建立

细胞核作为细胞中重要的遗传物质存储、复制和转录的结构,牵涉着大量信息和物质的传输活动,尤其是蛋白质的入核转运一直以来都是研究的热点问题之一。本文利用病毒SV40抗原蛋白中的核定位信号（nuclear localization signal,NLS）标记GFP蛋白,通过拟南芥细胞质的介导,利用HeLa细胞核建立起了研究蛋白质入核转运的半细胞体系。结果显示,植物细胞质结合NLS片段能改变GFP在HeLa细胞核内外的分布,实现对目标蛋白入核过程的介导,使GFP-NLS最后定位于细胞核内。这也意味着通过HeLa细胞建立起的半细胞体系能为蛋白入核转运研究提供一个有效的研究体系。